This invention is in the general field of biochemical engineering. More specifically, the subject invention relates to the preparation of human interferons and interferon-like polypeptides. Still more specifically, the subject invention relates to an improved process for the production and recovery of human fibroblast interferon and interferon-like polypeptides (.beta.-HIFN) from genetically transformed microorganisms, .beta.-HIFN preparations of relatively high purity, and therapeutically acceptable formulations thereof.
This application is also related to U.S. Ser. No. 353,360, "Process For Recovering Human IFN-.beta. From A Transformed Microorganism," filed March 1, 1982, and assigned to Cetus Corporation.
Naturally occurring interferons (IFNs) are species-specific proteins, often glycoproteins, produced by various cells upon induction with viruses, double stranded RNA's, other polynucleotides, antigens and mitogens. Interferons exhibit multiple biological activities such as antiviral, antiproliferative, immunomodulatory and anticellular functions. At least three distinct types of human interferons have been identified and characterized in terms of their anti-viral, anti-growth and activation of natural killer cell (NK) activities. They are produced by leukocytes, lymphocytes, fibroblasts and the immune system and are classified as .alpha., .beta. and .gamma. interferons. These are reported to be different proteins coded for by distinct structural genes.
Since their discovery in 1957, interferons have been extensively and intensively investigated for their potential therapeutic use as antiviral and/or anti-cancer agents. However, the paucity of adequate amounts of the material isolated from natural sources and the expense involved, have precluded extensive clinical testing and evaluation of the extent of the therapeutic value of these interferons.
In recent times, however, several of the human interferon genes have been cloned using recombinant DNA technology and expressed in E. coli (Nagola, S. et al., Nature 284: 316 (1980); Goeddel, D. V. et al., Nature, 287: 411 (1980); Yelverton, E., et al., Nuc. Acid Res., 9: 731 (1981); Streuli, M., et al., Proc. Nat'l. Acad. Sci. (U.S. ., 78: 2848 (1981). The expressed proteins or polypeptides have been purified and tested and have been found to exhibit properties similar to those of native IFNs. Bacterially produced IFN's thus appear to have potential therapeutic use as antiviral and anti-tumor agents and the production of IFN's by such bacterial fermentations is expected to yield large enough quantities of IFN at a relatively low cost for clinical testing.
Native .beta.-interferon is generally produced by superinducing human fibroblast cultures with poly-IC (poly-riboinosinic acid and polyribocytidylic acid) and isolating and purifying the .beta.-HIFN thus produced by chromatographic and electrophoretic techniques. Proteins or polypeptides which exhibit native .beta.-interferon like properties may also be produced using recombinant DNA (r-DNA) technology by extracting poly-A-rich 12S messenger RNA from virally induced human cells, synthesizing double-stranded c-DNA using the m-RNA as a template, introducing the c-DNA into an appropriate cloning vector, transforming suitable microorganisms with the vector, harvesting the bacteria and extracting the .beta.-HIFN therefrom. European Pat. application Nos. 28033, published May 6, 1981; 321134, published Jul. 15, 1981; 34307 published Aug. 26, 1981; Belgian Patent No. 837397, issued Jun. 1, 1981 describe various currently used methods for the production of .beta.-interferon employing r-DNA techniques.
However, interferon samples for use in clinical studies must be of relatively high purity and substantially uncontaminated with toxic host cell constituents, cell debris and other extraneous chemicals introduced during the extraction and purification steps. There are several methods currently available for the preparation, recovery and purification of bacterially produced IFNs.
U.S. Pat. No. 4,315,852, "Extraction of Interferon from Bacteria," issued to Leibowitz et al., describes and claims a method for the acid extraction of leukocyte interferon from bacterial cells and neutralization of the extractant to obtain the interferon.
Derynck et al., Nature, 287: 193 (1980) teach lysing transformed E. coli cells using a solution containing 5 M urea, 1% SDS, and 1% 2-mercaptoethanol. The lysate which was purified by chromatography, exhibited interferon activity.
Scandella and Kornberg, Biochemistry, 10: 4447 (1971) describe the preparation of a phospholipase from E. coli by solubilizing the cell membranes with SDS and precipitating the solubilized protein with 1-butanol.
U.S. Pat. No. 4,343,735, "Process For The Purification Of Interferon," issued to Menge, et al., teaches a process for the purification of interferon by partitioning it in an aqueous multi-phase system in the presence of ion exchangers which are soluble in the system and are derivatives of polyethers.
U. S. Pat. No. 4,343,736, "Process For Recovering Interferon," issued to Uemura et al., discloses a method for recovering interferon by absorption on water insolubilized heparin and then eluting the interferon with an aqueous solution of an inorganic salt and chondroitin sulfate.
A major problem with the above methods of purification and recovery of IFN is that the protein is not produced in a pure enough form and in large enough quantities for clinical and therapeutic purposes, and further, that the resulting IFN preparations, especially those that are produced by r-DNA techniques, have residual toxic amounts of chemicals, such as sodium dodecyl sulfate (SDS) and other surfactants or precipitants used in the extraction and purification steps. Thus these preparations are not acceptable for clinical studies designed to determine the extent of the therapeutic use and applications of IFN. It would be desirable, therefore, to have available a process for the recovery of .beta.-interferon in large enough quantities and without toxic levels of SDS for clinical and therapeutic applications.
Accordingly, it is an object of the present invention to provide a pharmaceutically acceptable sample of microbially produced .beta.-interferon which is of relatively high purity.
Another object of the present invention is to provide pharamaceutically acceptable samples of .beta.-interferon in sufficiently large quantities for clinical and therapeutic applications.
Yet another object of the instant invention is to provide .beta.-interferon preparations that are substantially free of SDS without loss of their biological activity, or at levels that are therapeutically acceptable.
A further object of this invention is to provide .beta.-interferon samples wherein the level of SDS is less than about 10 p.p.m.
Additional objects, advantages and novel features of the invention will be set forth in part in the description which follows, and in part will become apparent to those skilled in the art upon examination of the following or may be learned by practice of the invention. The objects and advantages may be attained by means of the instrumentalities and combinations particularly pointed out in the appended claims.